Bile resistant bacillus composition

ABSTRACT

A  bacillus  composition characterized by fast germination and outgrowth in bile salts (simulated gut environment) and by producing a compound of interest. The  bacillus  composition may be used as supplement in animal feed where it has a probiotic (health promoting) effect and increases the digestion and availability of nutrients from animal feeds.

FIELD OF THE INVENTION

The present invention relates to a bacillus composition characterized byfast germination and outgrowth in bile salts (simulated gut environment)and by producing a compound of interest. The bacillus composition may beused as supplement in animal feed where it has a probiotic (health andgrowth promoting) effect and increases the digestion and availability ofnutrients from animal feeds.

BACKGROUND ART

Probiotic bacteria such as Bacillus subtilis and Bacillus licheniformisare used in the animal feed industry as supplement to the diet. Theirusage is related to the ability of bacillus to replace or reduce the useof antibiotics, which are used as growth promoters in the animal feedindustry.

Christian Hansen A/S, Denmark commercializes an example of such aprobiotic growth-promoting product under the trade name GalliPro®(deposited as DSM 17231). GalliPro® is a Bacillus subtilis spore cellcomposition.

Besides the suggested mode of actions (e.g. immune modulation, gut floramodifier) probiotic bacillus are able to produce many beneficialcomponents, such as enzymes, which are excreted in the gastro intestinaltract (GIT) when used as animal feed supplement. Enzymes such as phytaseare excreted and improve the digestion and better uptake of animal feed(higher digestibility). The diet (feed) is mostly composed of plantorigin such as grains, corn, soybean, soy oil and amino acids. Overallthese effects contribute to the production of cost effective animalproducts.

Probiotic bacilli are also able to produce other beneficial componentssuch as essential amino acids.

Bacillus spores can pass the acidic gastric barrier and germinate andoutgrow within the gastrointestinal (GIT) of the animals. This has greatadvantages, since when ingested they can excrete numerous types ofbeneficial components, e.g. bacteriocins and also excrete usefulessential amino acids. Moreover, the bacillus spores are thermostabileduring a feed pelletizing process and are thereby an excellent deliverysystem to get both bacteriocins and e.g. essential amino acids into theGIT.

In the survival and proliferation process of bacillus in GIT, the roleof bile is important. Bile is produced in the liver and stored in thegallbladder. Bile contains water, lecithin, bilirubin and biliverdin andbile salts.

It is known from the literature that bile has some negative influenceson the survival and germination and outgrowth of bacillus spore cells tovegetative cells in the GIT of animals. Therefore research is ongoing tofind probiotic bile resistant Bacillus strains.

The article (Antonie Van Leeuwenhoek. 2006 August; 90(2): 139-46. Epub2006 Jul. 4) describes isolation of a number of Bacillus samples/celldirectly from the intestine of chickens. The isolated bacillus cellswere tested for probiotic activity. The six bacilli with highestprobiotic activity were testes for bile salt resistance and it was foundthat a specific highly probiotic bacillus has a relatively high level ofbile salt resistance.

In this article there is no special focus on any time periods for thetesting of bile resistance. In the experimental part the bacillus sporecells are simply tested for resistance after 5 days of presence in bilesalt (see paragraph “Simulated small intestinal fluid tolerance test” onpage 141).

US2003/0124104A describes that probiotic conventional bacillusendospores are sensitive to low concentration of bile salts, i.e. sporegermination and/or rehydration is inhibited by the presence of even lowconcentrations of bile salts. This is contrary to other bacteria such asenteric pathogens, such as E. coli or S. aureus (see section [0014] to[0015]). In view of this it is suggested to screen/select for bacillusspores that are resistant to the inhibitory activity of bile salts, andas a result, germinate into vegetative cells, which then colonize thecolon (see [0019]).

The working examples are all in presence and no real experimental dataof actually screened specific Bacillus cell are provided in thedescription.

Further the bile salt screening conditions are relatively genericallydescribed. In particular there are no indications of any time periodsfor the selections of bile resistance. Said in other words, based on theonly broad/generic teaching of this document one may select Bacilluscells that only can outgrow (germinate) slowly, i.e. are capable ofgerminating from spores to vegetative cells after e.g. 20 hours inpresence of relevant amount of bile salt.

In this document there is no description or suggestion to select forbacillus cells that can outgrow (germinate) rapidly, i.e. capable ofgerminating and outgrowing from spores to vegetative cells reaching adefined growth point within a certain time interval in presence of arelevant amount of bile salt.

In summary, the prior art references relating to selection/screening ofbile resistant bacillus cells are not focusing on rapidoutgrowth/germination from spore cells to vegetative bacillus cells.

International PCT application with application number PCT/EP2008/057296was filed Nov. 6, 2008. Applicant is Chr. Hansen A/S and it was NOTPUBLISHED at the filing date of this present application.

PCT/EP2008/057296 describes novel bacillus spores characterized byhaving an improved/rapid speed of germination and outgrowth from sporeto vegetative cell in presence of a bile salt medium.

The bacillus spores as described herein have the same improved/rapidspeed of germination and outgrowth from spore to vegetative cell asdescribed in PCT/EP2008/057296.

PCT/EP2008/057296 only describes bacillus vegetative cells that areproducing phytase in an increased amount as compared to the referencebacillus cell DSM 19467. As can be seen below, in first aspect and claim1 herein is disclaimed such high producing phytase bacillus cells ofPCT/EP2008/057296.

When there below is referred to prior art this shall be understood asprior art made available to the public (e.g. published articles/patents)at the filing date of this present application.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide abacillus composition which can excrete high amounts of beneficialcompounds in the gastro intestinal tract (GIT) of an animal.

The solution is based on that the present inventors have developed anovel selection method for the identification of new improved bacilluscompositions.

A novel important step of the herein described new selection method isto specifically screen/select for bacillus spore cells withimproved/rapid speed of germination and outgrowth from spores tovegetative cells in the presence of bile salts.

As described above, the prior art has described methods for selectingbacillus cells capable of growing in presence of bile salts, but theprior art screening/selection methods do NOT focus on the speed ofgermination and outgrowth in the presence of bile salt. Accordingly, theprior art selected bile resistant bacillus cells do not germinate andgrow fast enough to comply with the speed of germination and outgrowthcriteria as described herein. For instance, bacillus cells isolateddirectly from the intestine of e.g. chickens (as e.g. described in theAntonie Van Leeuwenhoek article discussed above) in the gut environmentare not selected (under natural pressure) to germinate and outgrowrapidly in the intestine.

As shown in working examples herein this is also true for the commercialavailable Bacillus composition GalliPro®, which simply germinates andoutgrows too slowly and does not reach the defined growth point withinthe first 20 hours in presence of physiological levels of bile salts tocomply with the speed of germination and out-growth criteria asdescribed herein. GalliPro® is a Bacillus subtilis composition that iscommercially successful.

The herein described novel DSM 19467 was selected by using GalliPro® asa starting strain and a selective pressure method and a subsequentisolation for rapid germination and outgrowth from spores to vegetativecells in presence of bile salt as described herein.

See e.g. table 1 for further details (GalliPro® may herein also betermed DSM 17231).

In FIG. 1 herein this is illustrated schematically.

In summary, it is believed that no prior art describes an isolatedBacillus composition, which comprises from 10⁵ to 10¹² CFU/g bacilluscells, wherein the cells of the bacillus composition complies with therapid germination and outgrowth in the presence of bile salt criteria asdescribed herein.

Without being limited to theory, the present inventors have identifiedthat rapid germination and outgrowth is a very important aspect of theinvention as bacillus spores, which are resistant to bile but do notgerminate and outgrow fast enough, will be excreted before any positivecharacteristics, such as essential amino acid production, can be made insignificant amounts by the vegetative bacillus cells. Bacillus sporesgerminating too slowly will simply pass through the gastro intestinaltract (GIT) before the bacteria can produce any significant amount ofe.g. essential amino acids.

After a number of detailed tests and analysis, the inventors thereforechose to work with a time range up to 20 hours and select the fastestgerminating and outgrowing spores within this time period in presence ofhigh physiological concentrations of bile salts. Without being limitedto theory and based on the herein disclosed detailed experimental work,the present inventors have identified that it is important to have arapid germination and outgrowth within the first 18 and 19 hours in thepresence of 4 and 6 mM bile salt, respectively.

The present inventors then identified that once bacillus cells, withrapid germination and outgrowth in bile salt medium, have been selectedthese cells are highly useful as starting cells for mutagenesis toobtain new cells with improved essential amino acid production.

As illustrated schematically in FIG. 1 and example 4, the rapidoutgrowing bile resistant selected strain, DSM 19467, was used asstarting strain for classical mutation and the high essential amino acidproducing strain were selected. As can be seen in example 4, some of theselected strains produce at least 5 times more of the essential aminoacid leucine than DSM 19467 and GalliPro®.

As discussed above, in our earlier application PCT/EP2008/057296 DSM19467 was use to make new high phytase producing strains—see forinstance example 4 of PCT/EP2008/057296.

The description of PCT/EP2008/057296 is incorporated herein byreference.

Accordingly, one may see DSM 19467 as a very useful kind of e.g.“intermediate” to make new bacillus cells with improved properties suchas e.g. higher phytase or essential amino acid production or any otherrelevant compound of interest.

Further, it is evident to the skilled person that once the inventorsherein have disclosed the ESSENTIAL test assay for testing rapidgermination and outgrowth of example 1 plus the identified strain DSM19467 it will be routine work for the skilled person to select other newbacillus cells complying with the criteria of the first aspectherein—i.e. other alternative bacillus cells with similar characteristicto DSM 19467.

Accordingly, a first aspect of the invention relates to a bacilluscomposition, which comprises from 10⁵ to 10¹² CFU/g bacillus sporecells, wherein the bacillus composition is characterized by:

(i): the bacillus spores have a rapid germination and outgrowth fromspore to vegetative cell in presence of a bile salt medium comprising 4and 6 mM bile salts, defined by that the bacillus spores reach avegetative cell growth point of 0.4 OD₆₃₀ within less than 18 and 19hours, respectively, wherein the vegetative cell growth point is thepoint in the growth curve where the OD value starts to increase (due togrowth of the vegetative cells) in a continuous way and reaches an OD₆₃₀of 0.4;

-   -   (I): wherein the bile salt medium is the standard known        non-selective Veal Infusion Broth (VIB) medium of example 1        herein supplemented with a bile salt mixture comprising the        conjugated bile salts taurodeoxycholate and glycodeoxycholate        and the deconjugated bile salt deoxycholate in the proportions        60% of the taurodeoxycholate, 30% of the glycodeoxycholate and        10% of deoxycholate; and    -   wherein the OD assay analysis is performed by the following        steps:    -   (a): filling a well in a microtiter plate with 0.150 ml bile        salt medium having 10⁸ bacillus spores per ml medium (i.e. this        is time zero); and    -   (b): incubating the plate at 37° C. under atmospheric conditions        and measuring the OD₆₃₀ values, using a spectrophotometer and        with agitation before each reading, to get a representative        growth curve over time; and

(ii) the bacillus vegetative cells are producing a relevant compound ofinterest;

and with the proviso that the bacillus vegetative cells are NOT cells asdescribed in claim 1 of PCT/EP2008/057296, wherein the bacillusvegetative cells of item (ii) are producing phytase in an amount of atleast 1.25 times more than the reference bacillus cell DSM 19467,wherein the produced phytase amount is measured by the standard phytaseassay of example 2 of PCT/EP2008/057296 after 4 hours growth at 37° C.in the standard known non-selective Heart Infusion Broth (HIB) medium ofexample 2 of PCT/EP2008/057296; and

wherein the phytase assay analysis is performed by the following steps:

-   -   (a): making a overnight culture of bacillus vegetative cells in        a enriched culture medium; and    -   (b): transferring a 1% inoculum from the overnight culture to        HIB medium (i.e. this is time zero) and incubation at 37° C.        until phytase activity measurement.

The term “a relevant compound of interest” of point (ii) should beunderstood broadly. As discussed above, bacillus cells with spores thatcomply with criteria (i) of first aspect can in principle e.g. be usedas a starting strain to screen for bacillus vegetative cells that maye.g. have higher/improved production of any compound of interest—such ase.g. an enzyme of interest.

As discussed above, the reference bacillus cell DSM 19467 is selectedfor rapid germination and outgrowth in presence of bile salt by usingGalliPro® as starting strain. DSM 19467 is not selected for improvedproduction of a compound of interest (e.g. an essential amino acid).Without being limited to theory, it is believed that the herein relevantproduction of the vast majority of commercial relevant compounds ofinterest (e.g. phytase or an essential amino acid) of DSM 19467corresponds to GalliPro® production of the same compound.

In relation to point (i) the vegetative cell growth point for GalliPro®is at least 20 hours after incubation in 4 and 6 mM bile salt and forthe novel DSM 19467 strain, as described herein, it is after 14 and 15hours in 4 and 6 mM bile salts, respectively (see FIG. 2 and workingexample 3 herein).

It is here relevant to note that the present inventors also tested thecommercial available product CALSPORIN® (Calpis Co., Ltd., Japan) todetermine the vegetative cell growth point under the conditions of point(i) of first aspect. As for GalliPro® the commercial product CALSPORIN®is a Bacillus subtilis composition used as a probiotic feed additive.The vegetative cell growth point under the conditions of point (i) offirst aspect for CALSPORIN® was more than 20 hours at 4 and 6 mM bilesalts, respectively. This is considerably more than the 18 and 19 hoursrequired under point (i) and this illustrates that commerciallyavailable products have hitherto not been selected for rapid germinationand outgrowth. As discussed above, “natural” bacillus cells have notbeen under any selective pressure to get rapid germination andoutgrowth. Without being limited to theory, it is therefore believedthat “natural” bacillus cells are not complying with the conditions ofpoint (i) of first aspect.

Both the bile resistance [of point (i)] and essential amino acid assay[of point (ii)] are based on known, commercially available standardelements (such as e.g. standard media, bile salts; standard ODmeasurements and standard tests).

The reference bacillus cell is deposited as DSM 19467 and is thereforepublicly available.

The Bacillus subtilis cell GalliPro® is deposited as DSM 17231 (named“GalliPro®”) and is therefore publicly available.

Accordingly, based on the detailed assay description herein (see e.g.example 1 herein for bile resistance assay and example 2 herein foressential amino acid assay) the skilled person is routinely able torepeat these assays to objectively determine whether a specific bacilluscell of interest complies with the bile resistance [of point (i)] andessential amino acid [of point (ii)] levels of the first aspect of theinvention.

The novel bacillus composition as described herein may be used as aprobiotic supplement to animal feed. The dose and administration may bedone according to the art as for instance as done for prior artGalliPro® bacillus compositions.

Accordingly, a second aspect of the invention relates to a method forfeeding an animal comprising administering the bacillus composition offirst aspect and herein described related embodiments to an animal inconjunction with other animal feed ingredients.

A third aspect of the invention relates to a method for screening andisolating a novel bacillus cell comprising the following steps:

-   -   (a): selecting and isolating from a pool of individual bacillus        spore cells of a new bacillus spore cell that is capable of        germinating and outgrowing so rapidly that it reaches a        vegetative cell growth point within less than 18 and 19 hours        under the conditions of point (i) of first aspect;    -   (b): making a vegetative bacillus cell from the isolated spore        cell of step (a) and mutating the novel selected and isolated        cell to get a pool of new individual bacillus vegetative cells;    -   (c): selecting and isolating from the pool of new individual        bacillus vegetative cells of step (b) a new bacillus vegetative        cell that is capable of producing a relevant compound of        interest; and    -   (d): analyzing the vegetative bacillus cell of step (c) to        confirm that it has maintained the rapid germination and        outgrowth of step (a) and isolating the selected bacillus cell.

It is evident to the skilled person that once the inventors herein havedisclosed the relevant test assays (in particular the assay for testingrapid germination and outgrowth of example 1) plus the reference strainDSM 19467 it will be routine work for the skilled person to select othernew bacillus cells complying with the criteria of the first aspectherein.

As discussed herein, by using the novel screening/selection method asdescribed herein the inventors have selected and isolated a number ofnew improved bacillus cells.

Embodiment of the present invention is described below, by way ofexamples only.

Definitions

All definitions of herein relevant terms are in accordance of what wouldbe understood by the skilled person in relation to the herein relevanttechnical context.

The term “bacillus cell” relates herein to both a bacillus spore celland a bacillus vegetative cell.

The term “bacillus spore” in relation to bacillus spore cell relatesherein to a spore that according to the art may be characterized as adormant, tough, non-reproductive structure produced by bacillusbacteria. The primary function of spores is generally to ensure thesurvival of a bacterium through periods of environmental stress. Theyare therefore resistant to ultraviolet and gamma radiation, desiccation,lysozyme, temperature, starvation, and chemical disinfectants. Sporesare commonly found in soil and water, where they may survive for longperiods of time. The spore coat is impermeable to many toxic moleculesand may also contain enzymes that are involved in germination. The corehas normal cell structures, such as DNA and ribosomes, but ismetabolically inactive. When a bacterium detects that environmentalconditions are becoming unfavorable it may start the process ofsporulation, which takes about eight hours.

The term “bacillus vegetative cell” relates to functional vegetativebacillus cells, which can divide to produce more vegetative cells.

The term “germination and outgrowth” relates to that bacillus sporesgerminate and outgrow to bacillus vegetative cells. As know to theskilled person reactivation of the spore occurs when conditions arefavorable and involves germination and outgrowth. Germination involvesthe dormant spore starting metabolic activity and thus breakinghibernation. It is commonly characterized by rupture or absorption ofthe spore coat, swelling of the spore, an increase in metabolicactivity, and loss of resistance to environmental stress. Outgrowthfollows germination and involves the core of the spore manufacturing newchemical components and exiting the old spore coat to develop into afunctional vegetative bacterial cell, which can divide to produce morecells.

Growth curves (OD versus time) of bacillus cells show distinct growthphases. As the spores are transferred to a nutrient rich medium thegermination is initiated followed by a temporary decrease in OD (phaseI), which is due to the release of dipicolinic acid and consequentlyhydration of the spore coat. In the second phase (phase II=outgrowthphase) there is a period with a relative little change in OD, until thespores are developed into a functional vegetative bacterial cells, whichcan divide to produce more cells and thereby give a continuous increasein OD value. The point when one starts to get the continuous increase inOD values reaching an OD of 0.4 is herein termed “vegetative cell growthpoint”.

The term “optical density” is defined as a measure of optical absorbanceusing a spectrophotometer. Optical density (OD) is the absorbance of anoptical element for a given wavelength A per unit distance. If OD ise.g. measured at wavelength 630 nm it may be referred to as OD₆₃₀.

DRAWINGS

FIG. 1: In this figure the steps to get to the herein novel improvedstrains are illustrated. The working examples herein were started fromDSM 17231 (GalliPro®) which was classically mutated andscreened/selected for rapid germination and outgrowth in presence ofbile salt to get the novel selected strain DSM 19467. DSM 19467 was usedas starting strain for classical mutation and high essential amino acidproducing strains were selected.

FIGS. 2 a and 2 b: These figures show clearly the improved rapidgermination and outgrowth of DSM 19467 bacillus spores of the presentinvention as compared to DSM 17231 in presence of 4 and 6 mM bile saltas described herein.

DETAILED DESCRIPTION OF THE INVENTION

A Relevant Compound of Interest:

As discussed above, the term “a relevant compound of interest” of point(ii) should be understood broadly. As discussed above, bacillus cellswith spores that comply with criteria (i) of first aspect—such as e.g.DSM 19467—can in principle e.g. be used as a starting strain to screenfor bacillus vegetative cells that may e.g. have higher/improvedproduction of any compound of interest—such as e.g. an enzyme ofinterest.

Suitable examples of a compound of interest may be enzymes such as e.g.mannanase. A compound of interest could also be an essential amino acidsuch as leucine.

In a preferred embodiment of point (ii) of the first aspect the bacillusvegetative cells are producing the compound of interest in an amountthat is higher than the reference bacillus cell DSM 19467, wherein boththe vegetative cell and reference bacillus cell DSM 19467 are grownunder identical conditions.

In example 4 herein on can see an example of this embodiment—in example4 herein is described vegetative cells with at least 5 times higherproduction of the essential amino acid leucine as compared to DSM 19467.

Further, as discussed above—in example 4 of PCT/EP2008/057296 isdescribed similarly for phytase—i.e. vegetative cells with higherproduction of phytase as compared to DSM 19467.

Bacillus Composition:

The term “bacillus composition” shall be understood according to theart. It is herein understood as a bacillus composition comprising anumber of bacillus spore cells with a characteristic of interest.

The bacillus composition may comprise different types of bacillus cells(e.g. B. subtilis and Bacillus licheniformis). In essence thecomposition shall simply comprise the amount of bacillus spore cellsgiven in the first aspect herein, wherein the bacillus cells comply withthe criteria given in the first aspect.

As known to the skilled person, herein commercially relevant bacillusspore cell compositions are generally made by fermentation. The obtainedspore cells are generally concentrated, dried, mixed with a carrier andpacked into a suitable container.

The relevant e.g. 10⁵ to 10¹² CFU/g bacillus cells of the compositionmay be present in a commercially relevant form known to the skilledperson.

Accordingly, in an embodiment 10⁵ to 10¹² CFU/g bacillus spore cells ofthe composition are present as dried (e.g. spray dried) cells or asfrozen spore cells.

In a preferred embodiment the bacillus composition comprises from 10⁶ to10¹² CFU/g bacillus spore cells, more preferably from 10⁷ to 10¹² CFU/gbacillus spore cells.

The term “CFU/g” relates to the gram weight of the composition as such,including suitable relevant additives present in the composition. Itdoes not include the weight of a suitable container used to package thebacillus composition.

An embodiment relates to that the bacillus composition is packaged intoa suitable container.

As known to the skilled person a commercially relevant bacterialcomposition generally also comprises other relevant additives such ase.g. one carrier/ingredient of the group belonging to whey, wheypermeate, calcium carbonate/limestone and anti caking agents such asaluminum silicates and kieselgur (diatomaceous earth).

Beside the herein relevant bacillus cells the composition may alsocomprise other relevant microorganisms of interest such as e.g. lacticacid bacteria of interest.

Bacillus Cell

The bacillus cell may be any relevant bacillus cell of interest.

In a preferred embodiment the bacillus cell is at least one bacilluscell selected from a bacillus species selected from the group consistingof:

Bacillus subtilis, Bacillus uniflagellatus, Bacillus lateropsorus,Bacillus laterosporus BOD, Bacillus megaterium, Bacillus polymyxa,Bacillus licheniformis, Bacillus pumilus, and Bacillussterothermophilus, Bacillus coagulans, Bacillus thermophilus, Bacillusmycoides, Bacillus cereus, and Bacillus circulars.

In a more preferred embodiment the bacillus cell is a B. subtilis cellor a Bacillus licheniformis cell.

The most preferred is wherein the bacillus cell is a B. subtilis cell.

Assay to Select for Rapid Germination and Outgrowth in the Presence ofBile Salt

As discussed above the bile resistance assay of point (i) of firstaspect is based on known commercially available standard elements (suchas e.g. standard media, bile salts; standard OD measurements).

Accordingly, based on the detailed assay description herein (see e.g.example 1 herein) the skilled person is routinely able to repeat thisassay to objectively determine whether a specific bacillus spore cell ofinterest complies with the rapid germination and outgrowth from spore tovegetative cell criteria as described in point (i).

In point (i) it is explained that vegetative cell growth point is thepoint in a growth curve starting with 10⁸ spores/ml corresponding to ODof around 0.2-0.3 until the time where the OD value has increased (dueto growth of the vegetative cells) in a continuous way and has reachedOD 0.4. This is in accordance with how a skilled person would understandsuch a vegetative cell growth point and based on a growth curve theskilled person may routinely determine this, within a limitedvariability of around ±30 minutes, as explained herein.

Working example 1 herein provides a detailed description of a bileresistance assay suitable to select for rapid germination and outgrowthin the presence of bile salt. The detailed conditions of this example 1is herein a preferred assay to determine if a bacillus spore cell ofinterest complies with the criteria of point (i) of first aspect.

The term “bile salt” relates to the salt of bile acids. Bile acids aresteroid acids found predominantly in the bile of mammals. They areproduced in the liver by the oxidation of cholesterol, and are stored ingallbladder and secreted into the intestine in the form of salts. Theyact as surfactants, emulsifying lipids and assisting with theirabsorption and digestion. The bile salts used in example 1 were preparedmimicking the physiological concentrations and compositions of porcinebile salts. As known to the skilled person porcine bile saltscompositions may herein be considered as relatively “harsh” conditionsas compared to avian bile salt compositions.

The term “bile salt medium” relates to medium comprising relevantbacillus growth ingredients such as relevant nutrients and bile salt.

Vegetative Cell Growth Point—in Bile Salt Assay—Point (i) of FirstAspect

As said above, in relation to point (i) of first aspect the bacillusspore cells, as described herein, have a germination and outgrowth fromspore to vegetative cell that is so rapid that they reach a vegetativecell growth point of 0.4 OD within less than 18 and 19 hours at 4 and 6mM bile salts, respectively.

As said above, the novel DSM 19467 strain reaches the vegetative cellgrowth point after 14 and 15 hours incubation in 4 and 6 mM bile salt,respectively.

Accordingly, in a preferred embodiment the bacillus spores reach thevegetative cell growth point after 17 and 18 hours incubation in 4 and 6mM bile salt under the conditions of point (i) of first aspect, morepreferably the bacillus spores reach the vegetative cell growth pointafter 15 and 16 hours incubation in 4 and 6 mM bile salt under theconditions of point (i) of first aspect.

As explained above and shown schematically in FIG. 1 the hereindescribed novel DSM 19467 strain was selected by using the commerciallyavailable GalliPro® as a starting strain for mutagenesis and selectionfor rapid outgrowth in presence of bile salt as described herein.

GalliPro® is a composition comprising Bacillus subtilis cells and theBacillus subtilis is deposited as DSM 17231. Accordingly, GalliPro® mayherein be seen as a reference strain.

As said above, the vegetative cell growth starting point for GalliPro®is after 20 hours incubation in 4 and 6 mM bile salts under theconditions of point (i) of first aspect. Accordingly, in an embodimentthe bacillus spores reach the vegetative cell growth point at least 3hours earlier than the reference Bacillus subtilis spores cellsdeposited as DSM 17231 (“GalliPro®”) under the conditions of point (i)of first aspect, more preferably the bacillus spores reach thevegetative cell growth point at least 4 hours earlier than the referenceBacillus subtilis spores cells deposited as DSM 17231 (“GalliPro®”)under the conditions of point (i) of first aspect, and most preferablythe bacillus spores reach the vegetative cell growth starting point atleast 5 hours earlier than the reference Bacillus subtilis spores cellsdeposited as DSM 17231 (“GalliPro®”) under the conditions of point (i)of first aspect.

Essential Amino Acids

As known to the skilled person an essential amino acid may be anessential amino acid selected from the group consisting of:phenylalanine, valine, threonine, tryptophan, isoleucine, methionine,leucine, lysine, cysteine, tyrosine, histidine and arginine.

In a preferred embodiment the essential amino acid is at least oneessential amino acid selected from the group consisting of:phenylalanine, valine, threonine, tryptophan, isoleucine, methionine,leucine, and lysine.

In more preferred embodiment the essential amino acid is at least oneessential amino acid selected from the group consisting of: valine,isoleucine and leucine.

A herein very relevant essential amino is leucine.

As understood by the skilled person, the bacillus vegetative cells mayproduce higher amount of more than one essential amino acid, such ase.g. higher amount of two or three or more different essential aminoacids.

Amino Acid Assay

As discussed above the amino acid assay of point (ii) of first aspect isbased on standard known commercially available elements (such as e.g.standard media, standard test).

Accordingly, based on the detailed assay description herein (see e.g.example 2 herein) the skilled person is routinely able to repeat thisassay to objectively determine whether a specific bacillus vegetativecell of interest complies with the produced essential amino acid amountas described in point (ii).

Working example 2 herein provides a detailed description of a essentialamino acid assay.

The detailed conditions of this example 2 are herein a preferredessential amino acid assay to determine if a bacillus vegetative cell ofinterest complies with the criteria of point (ii) of first aspect.

Produced Amount of Essential Amino Acid—Point (ii) of First Aspect

In relation to point (ii) of first aspect, the Bacillus vegetative cellsare preferably producing at least one essential amino acid in an amountof at least 2 times more than the reference Bacillus cell DSM 19467under the conditions of point (ii) of first aspect.

In a more preferred embodiment in relation to point (ii) of firstaspect, the Bacillus vegetative cells are preferably producing at leastone essential amino acid in an amount of at least 4 times more than thereference Bacillus cell DSM 19467 under the conditions of point (ii) offirst aspect.

A Method for Feeding/Administering Bacillus Spores to an Animal

As said above a second aspect of the invention relates to a method forfeeding an animal comprising administering the bacillus composition offirst aspect and herein described related embodiments to an animal inconjunction with other animal feed ingredients.

The animal may be any animal of interest. Preferably, the animal is ananimal selected from the group consisting of poultry, ruminants, calves,pigs, rabbits, horses, fish and pets.

When administering GalliPro® according to the art it is normally done ina dose from around 10⁴-10⁸ CFU/g feed, commonly 10⁵-10⁶ CFU/g feed or indoses equivalent to normal feed intake/kg live weight animal.

Alternatively the bacillus spores may be administered to the animal inone of the following ways:

(1): put it into drinking water for animals;

(2): sprayed onto animals; or

(3): application via paste, gel or bolus.

A Method for Screening and Isolating a Novel Bacillus Cell

As said above, the third aspect relates to a method for screening andisolating a novel bacillus cell.

In the method of the third aspect is selected for a bacillus cellcapable of fulfilling the conditions of point (i) and (ii) of the firstaspect.

As understood by the skilled person, the specific herein detaileddescribed bile resistance and essential amino acid amount assay (seee.g. example 1 herein for bile resistance assay and example 2 herein foressential amino acid assay) parameters may be changed to make aalternative screening method that still obtains the main goals asdescribed herein, i.e. a bacillus cell that is capable of fulfilling theconditions of point (i) and (ii) of the first aspect.

In a preferred embodiment, bile resistance assay of example 1 is used instep (a) of the screening method of third aspect and the essential aminoacid assay of example 2 is used in step (c) of the screening method ofthird aspect.

In step (d) of the screening method of third aspect a vegetativebacillus cell is isolated. This vegetative bacillus cell may be used tomake bacillus spores from.

Accordingly, in an embodiment the screening method of third aspect isfollowed by a extra step (e), wherein the isolated bacillus vegetativecell of step (d) is fermented to make from 10⁵ to 10¹² bacillusvegetative cells and these 10⁵ to 10¹² bacillus vegetative cells areused to make 10⁵ to 10¹² bacillus spore cells, which are isolated togive a Bacillus composition, which comprises from 10⁵ to 10¹² CFU/gbacillus spore cells.

The end result of step (e) is a novel Bacillus composition, whichcomprises from 10⁵ to 10¹² CFU/g bacillus spore cells, and wherein thebacillus cells are capable of fulfilling the conditions of point (i) and(ii) of the first aspect.

Accordingly, a separate aspect of the invention relates to a Bacilluscomposition, which comprises from 10⁵ to 10¹² CFU/g bacillus sporecells, and wherein the bacillus cells are capable of fulfilling theconditions of point (i) and (ii) of the first aspect obtainable by thescreening method of third aspect followed by extra step (f) describedabove.

In step (b) of the screening method of third aspect is made mutations ofthe earlier selected bile resistant bacillus cell to select for highessential amino acid producing cells in step (c). As understood by theskilled person this may e.g. by classical mutation (e.g. by chemicaltreatments or UV) of specific exchange of genes to make a so-calledGenetic Modified Organism (GMO).

Deposited Strains

A sample of the novel Bacillus subtilis strain has been deposited atDSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Maschroder Weg 1b, D-38124 Braunschweig) under the accession number DSM19467 with a deposit date of Jun. 27, 2007. The deposit has been madeunder the conditions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.

EXAMPLES Example 1 Bile Resistance Assay

Medium:

The medium was a standard non-selective commercial available medium VealInfusion Broth (VIB) (Difco, 234420).

At the filing date of the present application the product catalogue(“Difco™/BBL™ Manual) from the provider BD Diagnostic Systems(www.bd.com) read in relation to the Veal Infusion Broth:

“Infusion from lean veal and peptone provide the nitrogen, vitamins,carbon and amino acids in veal infusion media. Sodium chloride maintainsthe osmotic balance of the formulations”; and

The medium was prepared according to manufacture instructions bysuspending 25 g of the Veal Infusion Broth powder in 1 L of purifiedwater (2.5% solution) and heat with frequent agitation and boil for 1minute to completely dissolve the powder.

A 2.5% Veal Infusion Broth solution comprised per liter:

Lean Veal, Infusion: 10 g

Proteose Peptone: 10 g

Sodium Chloride 5 g

The medium was distributed into sterile bottles and autoclaved for 15min at 121° C.

Bile Salt Solutions/Medium:

Mixtures of bile salts were prepared mimicking the physiologicalcomposition and concentration of bile salts in pig bile and the bilesalts were dissolved in the Veal Infusion Broth medium as prepared aboveto give a final bile salt concentration of 8 mM.

The conjugated bile salts were taurodeoxycholate (Sigma T-0875, U.S.)and glycodeoxycholate (Sigma G-9910, U.S.) and the deconjugated bilesalt deoxycholate (Sigma D-5670 U.S.) and the final 8 mM mixed bile saltsolution contained 60% of the taurodeoxycholate, 30% of theglycodeoxycholate and 10% of deoxycholate. Before autoclaving for 15minutes at 121° C., the solutions were adjusted to pH 7.4 using sodiumhydroxide. The prepared 8 mM bile salt medium, were diluted to get bilesalt concentrations of 0, 1, 2, 4, 6 and 8 mM.

The bile salts were added to the Veal Infusion Broth medium in aconcentrated form.

Accordingly, the final amount of lean veal infusion, Proteose Peptoneand Sodium chloride were essentially as for the 2.5% Veal Infusion Brothmedium before the bile salts were added.

Spore Suspensions

To distinguish between vegetative cells and spores and to ensure purespore products for inoculation, the spore counts of the bacillus productwere determined using +/−heat treatment at 80° C. for 10 min. After heattreatment and subsequent cooling to room temperature, serial 10-folddilutions were conducted in saline peptone water. Duplicates of TryptoseBlood Agar plates (Difco 0232-01) were inoculated with 0.1 ml from theappropriate decimal dilutions. The plates were incubated at 37° C. untilthe next day. Based on preceding spore count determinations of theproducts, spore suspensions were prepared in sterile distilled water toreach final calculated spore concentration of 10⁸ CFU/ml. The counts ofvegetative cells and spores in the final inocula were determined usingthe method described above. The final concentration of 10⁸ CFU/mlcorresponded to a start OD₆₃₀ at 0.2-0.3.

Growth Measurement: Optical Density Measurements

Sterile flat bottom 96 well microtiter plates were used (Greiner Bio-oneGmbH, Germany). Each well was filled with 0.150 ml VIB inoculated withspores (˜1×10⁸ spores per ml equivalent/corresponding to a startOD₆₃₀˜0.2-0.3) and the plates were incubated for 20 hours at 37° C. witha 1 minute shaking cycle of intensity 4 (high) before each reading.

To avoid condensation on the inside of the plate cover, the lids wereexposed to a dilute solution of Triton X-100.

The germination and outgrowth kinetics of Bacillus strains were measuredusing a spectrophotometer at wavelength 630 nm (OD₆₃₀) (Bio-tekInstruments, Inc. VE). Readings were performed with 10 minute intervalsand analyzed using the KC4™ software (Bio-tek Instruments, Inc., USA).After 20 h, data were exported to Excel® spreadsheets for furtheranalysis, imported in SAS version 9.0 and statistically analyzed.

Example 2 Amino Acid Assay

The method to measure and quantify the amino acids produced by thebacillus cells used in this study is a standard GC-MS method for aqueoussamples, using methyl chloroformate as derivatization agent.

Growth of Bacillus Cells

The Bacillus cells are inoculated and grown in a minimal salts growthmedium at 37° C., 150 rpm and grown for 2 days and amount of amino acidis then measured in the supernatant as described below.

The bacillus cells are propagated in a Minimal Salts Medium according toChapman (1972) with the following composition:

(NH₄)₂SO₄ (Merck 1.01217.1000) 1 g/l K₂HPO₄ (Merck 1.05101.1000) 7 g/lKH₂PO₄ (Merck 1.04873.1000) 3 g/l MgSO₄•7H₂O (Merck 1.05886.1000) 0.1g/l  

Autoclaved for 15 min at 121° C. and added autoclaved glucose to a finalconcentration of 0.5%.

Incubation is done in tubes with 10 ml medium for 2 days at 37° C. and150 rpm.

Amino Acid Assay

The amino acid assay is carried out on cell supernatants, since theamino acids are secreted to the media. Samples are sterile filtered andkept at −20° C. until analysis.

Reagents:

Reagent 1: Internal standard solution. Norvaline 1 mM: 0.0172 gNorvaline+100 ml MQW

Reagent 2: Methanol/Pyridine 32/8 (v/v) (Catalysator)

Reagent 3: Methyl Chloroformate p.a. (MCF) (Derivatization agent)

Reagent 4: 1% MCF/CHCl₃ (v/v) (Extraction): 1 ml Methyl Chloroformatep.a.+Chloroform ad 1000 ml.

Sample Preparation:

-   -   Pipette 150 μl (25 μl+125μ MQW) sample into 2 ml injection vial.    -   Add 150 μl IS    -   Add 200 μl 1-Methanol/Pyridine 32/8% (v/v). Mix well.    -   Add 25 μl MCF (Methyl Chloroformate). Mix well until gas        development occurs.    -   Add 500 μl 1% MCF/CHCl₃ (v/v), cap and mix vigorously. Phase        separation occurs within minutes. If phase separation is too        slow, centrifuge the vial (500 rpm/10 min).

If Norvaline is used as antimetabolite, an external standard or anothersuitable internal standard should be used instead, and the 150 μl ISsubstituted with either MQW or sample.

Samples are run on GC-MS with a standard amino acid column and protocol.

Example 3 Selection of Bile Resistant Bacillus subtilis Cell DSM 19467

The starting bacillus cell was the Bacillus subtilis cell GalliPro®.

GalliPro® was mutagenized to get a pool of new individual bacilluscells. Spores were made and selected for rapid germination and outgrowthfrom spore to vegetative cell in presence of a bile salt mediumcomprising 4 and 6 mM bile salt a described in example 1 above.

Bacillus subtilis cell DSM 19467 was selected.

Table 1 below shows germination and outgrowth data.

Time (hours) from 10⁸ CFU/ml corresponding to OD 0.2-0.3 until OD 0.4 isreached (mean of 3 replicates).

B. subtilis 4 mM bile 6 mM bile Existing product GalliPro ® >20 >20 (DSM17231) Bile tolerant 13 h 40 m 15 h (DSM 19467) Commercial product:Calsporin >20 >20

Some of the data of this example was made by testing phytaseoverexpressing DSM 19489. But for the technical result of this examplethis is herein relatively irrelevant since DSM 19467 has germination andoutgrowth roughly as DSM 19489. See PCT/EP2008/057296 for furtherdetails.

Conclusion

DSM 19467 is a bile resistant strain and clearly germinating andoutgrowing faster than GalliPro®.

Example 4 Selection of Amino Acid Over-Producing Bacillus Cells from DSM19467

The starting bacillus cell was the Bacillus subtilis cell DSM 19467selected in example 3.

DSM 19467, either wildtype or mutants produced by, e.g., UV-mutagenesis,was grown on Minimal Salts Medium agar, described in example 2B aboveand added 1.5% agar, containing amino acid analogues in suitableinhibitory amounts. Depending on the amino acid to be over-expressedvarious amino acid analogues could be used, e.g., norvaline or4-aza-DL-leucine for overproducing leucine (Bardos, 1974, Topics inCurrent Chemistry 52, 63-98). Colonies resistant to the amino acidanalogue were picked, grown in Minimal Salts Medium and assayed foramino acid production. The vegetative cells were selected for producinghigh amount of amino acid by using the GC-MS method described in example2B above.

High amino acid producing Bacillus subtilis cell was selected.

Results of Amino Acid Measurements

A number of strains were selected which were producing the essentialamino acid leucine in an amount that was significant higher than thereference bacillus cell DSM 19467.

A number of the selected strains produced at least 5 times more leucinethan DSM 19467.

Conclusions:

This example shows that one can routinely—based on the instructionsherein—screen and identify a strain, which produces at least oneessential amino acid (here exemplified by leucine) in an amount that wassignificant higher than the reference bacillus cell DSM 19467.

DSM 19467 is originating from GalliPro® and is not selected for highessential amino acid production. Accordingly, it is believed thatGalliPro® produces roughly the same amount of essential amino acid asDSM 19467.

Example 5 Bile Resistance “Check” of High Essential Amino Acid ProducingBacillus Cells

Preferred high essential amino acid producing bacillus cells selected inexample 4 are re-checked for their ability of rapid germination andoutgrowth from spore to vegetative cells as described in example 1.

The results are that they—as expected—have maintained roughly the samegood rapid germination and outgrowth as the starting cell DSM 19467 usedto obtain them.

REFERENCES

1. Antonie Van Leeuwenhoek. 2006 August; 90(2):139-46. Epub 2006 July 4

2. US2003/0124104A

3. US6255098

4. PCT/EP2008/057296

1-12. (canceled)
 13. A Bacillus composition comprising from 10⁵ to 10¹²CFU/g Bacillus spore cells of a mutant Bacillus strain, wherein: (a) theBacillus spores have a germination and outgrowth from spore tovegetative cell in the presence of a bile salt medium comprising 4 mM or6 mM bile salt such that the spores reach a vegetative cell growth pointof 0.4 OD630 within less than 18 hours or 19 hours, respectively; and(b) the Bacillus vegetative cells overproduce at least one desiredcompound in comparison to the wild-type Bacillus strain from which themutant Bacillus strain derives when grown under comparable conditions.14. The Bacillus composition of claim 13, wherein the desired compoundis an enzyme.
 15. The Bacillus composition of claim 14, wherein theenzyme is phytase or mannanase.
 16. The Bacillus composition of claim13, wherein the desired compound is an amino acid.
 17. The Bacilluscomposition of claim 16, wherein the amino acid is selected from thegroup consisting of phenylalanine, valine, threonine, tryptophan,isoleucine, methionine, leucine, lysine, cysteine, tyrosine, histidine,and arginine.
 18. The Bacillus composition of claim 13, wherein theBacillus spore cells are present as dried spore cells.
 19. The Bacilluscomposition of claim 13, wherein the Bacillus is Bacillus subtilis. 20.A method for isolating a mutant Bacillus strain which germinates andoutgrows rapidly in the presence of a bile salt and which overproducesat least one desired compound, comprising: (a) subjecting a wild-typeBacillus spore cell to mutagenesis to isolate a Bacillus spore cellhaving a germination and outgrowth from spore to vegetative cell in thepresence of a bile salt medium comprising 4 mM or 6 mM bile salt suchthat the Bacillus spores reach a vegetative cell growth point of 0.4OD630 within less than 18 hours or 19 hours; (b) subjecting vegetativecells grown from the isolated spore cell of step (a) to mutagenesis,thereby obtaining a pool of individual vegetative cells which retainrapid germination and outgrowth in the presence of a bile salt; and (c)selecting a vegetative cell from the pool of step (b), which vegetativecell retains rapid germination and outgrowth in the presence of a bilesalt, and which overproduces the at least one desired compound.
 21. Themethod of claim 20, wherein the Bacillus strain is Bacillus subtilis.22. A method for feeding an animal comprising administering the Bacilluscomposition of claim 1 to the animal.